For a purpose such as the regulation of the properties of a biopolymer, the biopolymer has conventionally been conjugated with a non-peptide hydrophilic polymer such as polyethylene glycol (PEG) (herein after may be referred to as “conjugation”, or “pegylation” when PEG or its similar compound is used). More specifically, conjugation is carried out generally by binding an active group to the terminal of a non-peptide hydrophilic polymer and then reacting the active group with a functional group present on the molecular surface of a protein or the like.
Particularly, the conjugation of a protein or a peptide is important, and the partial coverage of the molecular surface of a protein with a chain of a non-peptide hydrophilic polymer has been studied for shielding an epitope of the protein to reduce the antigenicity and immunogenicity thereof, for reducing the incorporation thereof into the reticuloendothelial system etc., or for preventing the recognition and degradation thereof by proteases. It is also known that the in vivo clearance of such complex substance is delayed to prolong its in vivo lifetime. On the other hand, it is frequently observed that an active site of such complex protein or the like is affected by the presence of the non-peptide hydrophilic polymer to result in reduced biological activity.
For example, interferon when complexed with PEG prolongs its in vivo lifetime about 70-fold but reduces its biological activity such as antiviral activity to about 1/10. From a comprehensive viewpoint, however, the conjugation of interferon with PEG is known to result in significant improvement in its therapeutic effect and is useful for the treatment against hepatitis C.
In the concept of protein conjugation, there has been a long history since the successful conjugation of asparaginase with PEG for use of this enzyme as a drug for leukemia. Until now, the structures of conjugating reagents such as PEG (type of their active group, the size and distribution of their molecule, development of branched type, etc.) have been improved and the technologies are advancing.
Complexes of certain proteins with branched PEG are known to have higher protease resistance than its counterpart complexes with linear PEG, and to have increased stability against pH and heat depending on the protein (Non-patent Document 1: Monfardini et al., Bioconjug. Chem. 1995 6(1): 62-9). As to interferon, a complex thereof with branched PEG has a higher antiproliferative activity than that of its counterpart with other PEG or that of the interferon itself (Patent Document 1: Japanese Patent Application Laid-Open No. H10-67800).
However, a fluctuation in the activity of individual proteins upon conjugation will vary from protein to protein. Further, conjugation of a certain protein with PEG can bring about various influences on plural properties of the protein; for example, conjugation of interferon with PEG causes a decrease in its in vitro antiviral activity and an increase in its antiproliferative activity in human tumor cells. Accordingly, the optimum conditions and the like for obtaining a complex endowed with desired properties should be sufficiently examined for each protein.
It can be easily anticipated that depending on the structure of a chain (linear or branched chain, molecular size, distribution and so on) of a non-peptide hydrophilic polymer, the reaction sites and the number of reacting molecules, the conjugation of proteins, and so on exerts various influences on biochemical and pharmaceutical properties such as antigenicity, protease resistance, in vivo lifetime and heat stability, and on biological activities involved in drug efficacy. Accordingly, when such complexes are to be developed as pharmaceutical preparations, a non-peptide hydrophilic polymer chain should be added at a certain site or sites in order to guarantee predetermined qualities.
Lactoferrin (hereinafter abbreviated sometimes to “LF”) is a glycoprotein with a molecular weight of about 80,000 occurring mainly in mammalian milk and also found in neutrophils, tears, saliva, nasal discharge, bile, semen and so on. Lactoferrin binds iron and thus belongs to the transferrin family. Known physiological activities of lactoferrin include an antibacterial action, an iron metal metabolism regulating action, a cell growth activating action, a hematopoietic action, an anti-inflammatory action, an antioxidant action, a phagocytosis promoting action, an antiviral action, a bifidobacteria growth promoting action, an anticancer action, a cancer metastasis inhibiting action and a translocation inhibiting action. Recently, lactoferrin has also been revealed to have a lipid metabolism improving action, an analgesic/antistress action and an anti-aging action. As described above, lactoferrin is a multifunctional bioactive protein showing various functions and is expected for use in pharmaceutical preparations and foods for restoration or promotion of health, and lactoferrin-containing foods have already been commercially available.
Lactoferrin, when orally ingested, undergoes hydrolysis by an acid protease, pepsin, occurring in gastric juice thereby being decomposed into peptides, and thus hardly arrives as the lactoferrin molecule at the intestinal tract. In the gastrointestinal tract, however, lactoferrin receptors are known to occur in the mucosa of small intestine, and it has recently been revealed that lactoferrin is incorporated via the intestinal tract into the body, to express its bioactivity. Therefore, for exhibiting the bioactivity of lactoferrin, it is important that lactoferrin is allowed to arrive at the intestinal tract without undergoing hydrolysis by pepsin in the gastric juice.
With respect to lactoferrin, there is also a report on its PEG complex (Non-patent Document 2: C. O. Beauchamp et al., Anal. Biochem. 131: 25-33 (1983)). However, this literature merely describes that a complex of LF with linear PEG has an in vivo lifetime prolonged 5- to 20-fold, and does not describe the bioactivity of pegylated LF or the degree and uniformity of pegylation.    Patent Document 1: Official gazette of Japanese Patent Application Laid-Open No. H10-67800    Non-patent Document 1: Monfardini et al., Bioconjug. Chem. 1995 6(1): 62-9    Non-patent Document 2: C. O. Beauchamp et al., Anal. Biochem. 131: 25-33 (1983)